Compositions comprising viruses and methods for concentrating virus preparations

ABSTRACT

A composition is disclosed comprising virus in a formulation comprising a polyhydroxy hydrocarbon buffered to maintain a pH in a range from about 7 to about 8.5 at a temperature in the range from about 2° C. to 27° C. Methods for concentrating and purifying virus preparations are also disclosed.

I. FIELD OF THE INVENTION

The present invention relates to compositions comprising viruses,especially viral vectors, having significantly improved stability. Thecompositions of the present invention are useful in maintaining thestability of viruses during storage, and virus-containing compositionsof the present invention are particularly useful for therapeutic usessuch as gene therapy. New methods for concentrating and purifying viruspreparations are also provided.

II. BACKGROUND

Viruses have become increasingly important for therapeutic uses, such asvaccinations and gene therapy, and there is a need to develop andprepare stable virus-containing compositions that can easily be storedand transported, yet retain sufficient safety and efficacy. Inparticular, given the extensive use of viral vectors in gene therapy, itis important to develop and prepare formulations that can stablypreserve live recombinant viruses when they carry therapeutictransgenes.

Moreover, there is a critical need for formulations that can stabilizeviral preparations at temperatures above −80° C. for extended periods oftime. Virus-containing compositions normally require storage at −80° C.and cannot be stored at standard refrigeration temperatures (e.g., 2° C.to 8° C., or higher) for substantial periods of time. This limitationrepresents a serious impediment not only to storage, but also toprocessing, distribution, and widespread clinical use.

There is also a need to develop virus-containing compositions that canmaintain pH in the range of about 7 to about 8.5 for extended periodsdespite being exposed to refrigeration temperatures, and despite beingsubjected to harsh conditions such as freeze/thaw, especially the slowrates of freeze/thaw that can occur in connection with larger scaleproduction, handling, or distribution. Maintenance of pH is importantfor viral preparations because at pH below 7.0 and above 8.5 the livevirus particles are vulnerable to losing viability due to physical andbiological instability.

Additional problems relate to increasing virus concentrations. Inparticular, high virus concentration contributes significantly to virusinstability. However, increasingly higher concentrations of virus andviral vectors are required for therapeutic use. Therefore, there is acritical need to develop formulations that stabilize relatively highconcentrations of virus, especially under the harsh conditions mentionedabove. And in addition, there is a particular need to develop newmethods of concentrating an existing virus preparation to achieve stablepreparations at higher concentration levels. The problems of instabilityassociated with higher virus concentrations are exacerbatedsignificantly if one tries to concentrate an existing virus preparation.This is in part due to the additional mechanical shear forces that cometo bear during efforts to increase the concentration of an existingvirus preparation. If one could find a method to concentrate a viruspreparation without substantial impairment to virus stability, thenclinical dosages at any desired concentration could be readily prepared(even when starting with material having a lower concentration) and,importantly, the ability to concentrate virus could eliminateproblematic bottlenecks and other scale-up problems during thepurification process by allowing significantly higher throughput duringvarious processing steps such as size exclusion chromatography.

There is thus a need for materials and methods to accomplish theforegoing objectives.

III. SUMMARY OF INVENTION

The present invention fills the above-mentioned needs by providing astable composition comprising virus in a formulation comprising apolyhydroxy hydrocarbon buffered to maintain a pH in a range from about7 to about 8.5 at a temperature in the range from about 2° C. to 27° C.

Also provided are new methods of concentrating an existing viruspreparation that allow one to readily select and prepare clinicaldosages in a wide range of desired concentrations. A preferred method ofconcentrating a virus preparation comprises:

-   -   (a) adding a polyhydroxy hydrocarbon to a virus preparation to a        final polyhydroxy hydrocarbon concentration of about 20% or        more; and    -   (b) subjecting the virus preparation to a filtration process        wherein the concentration of virus is increased by applying        pressure to the preparation such that diluent is removed from        the virus preparation through a filter while the virus is        retained.        Also provided herein is a method for concentrating a virus        preparation comprising:    -   (a) centrifuging a composition which comprises a first layer        comprising a polyhydroxy hydrocarbon in a concentration of 35%        to 80% (v/v), the first layer overlaid with a second layer        comprising a polyhydroxy hydrocarbon in a concentration of 5% to        30% (v/v), the second layer overlaid with a third layer        comprising virus; and    -   (b) recovering the virus from the first layer.

Furthermore, the present inventors found that their new method ofincreasing virus concentration has the additional advantage of enhancingfurther processing (e.g. by reducing or eliminating problematicbottlenecks during subsequent purification by allowing significantlyhigher throughput during processing steps such as size exclusionchromatography). Thus, in a preferred embodiment, the method ofconcentrating virus preparations in accordance with present inventionfurther comprises a subsequent purification step (e.g., size exclusionchromatography). In this regard, the method of the present invention isparticularly useful when a step of size exclusion chromatography isperformed subsequent to ion exchange chromatography, and the viruspreparation is concentrated (in accordance with the present invention)after the ion exchange chromatography but prior to the size exclusionchromatography. Viral fractions obtained from anion exchangechromatography, for example, typically contain high levels of salts andpossibly other impurities that further compromise virus stability duringconcentration procedures. Thus, in a particularly preferred embodiment,the present invention provides a method of purifying a virus preparationcomprising:

-   -   (a) subjecting the virus preparation to anion-exchange        chromatography, wherein the virus is eluted as a virus        preparation product from an anion-exchange chromatographic        medium;    -   (b) adding a polyhydroxy hydrocarbon to the virus preparation        product of step (a) so that the concentration of polyhydroxy        hydrocarbon in the preparation reaches a final concentration of        about 25% or more; and    -   (c) increasing the concentration of virus in the virus        preparation product of step (b) by applying pressure to the        preparation such that diluent is removed from the virus        preparation through a filter while the virus is retained; and    -   (d) subjecting the concentrated virus preparation product of        step (c) to one or more additional processing steps.

The present invention also provides virus preparations concentratedand/or purified by the foregoing methods.

IV. DETAILED DESCRIPTION

As noted above, the present application discloses novel virus-containingcompositions, as well as novel methods of concentrating and purifyingvirus-containing compositions.

With regard to compositions, the present inventors have developed anovel buffered formulation that can preserve viral preparations withenhanced stability. In particular, the formulation can stabilize viralpreparations at temperatures well above −80° C. More important still,the compositions of the present invention are stable at typicalrefrigeration temperatures of, e.g., 2° to 8° C., or higher, forsubstantial periods of time, preferably for several months or more. Thisis a critical advantage because, as mentioned above, in order to meetclinical needs it is impractical to keep viral preparations frozen at−80° C. during storage and transportation.

An important feature of the compositions of the present invention is theaddition of a polyhydroxy hydrocarbon. As used herein, a polyhydroxyhydrocarbon means a branched, linear, or cyclic compound substitutedwith 2 or more (preferably 2 to 6, more preferably 2 to 4) hydroxygroups. Polyhydroxy hydrocarbons for use in the present inventionpreferably are polyhydroxy-substituted alkyl compounds (branched orunbranched), preferably having 2 to 7 carbon atoms, and can include,e.g., glycerol, sorbitol and polypropanol. Glycerol is particularlypreferred. As shown by data provided below, the present inventors foundthat glycerol allows for surprisingly high levels of stability forextended periods of time even under standard refrigeration conditions.

An effective amount of polyhydroxy hydrocarbon for compositions of thepresent invention is an amount sufficient to stabilize the virus in theformulation of the present invention without adversely affecting theeffectiveness of the virus for further use, especially in cases wherethe virus contains a transgene for use in gene therapy. The polyhydroxyhydrocarbon is preferably present at a final concentration of about 20to 200 mg/mL. A narrower range can be 80 to 120 mg/mL. More than onepolyhydroxy hydrocarbon can be used to achieve the desired total amountof polyhydroxy hydrocarbon in the composition of the present invention.

The polyhydroxy hydrocarbon in compositions of the present invention canoptionally contain an aldehyde group. In particular, the polyhydroxyhydrocarbon can be a disaccharide such as sucrose. Furthermore, even ifthe polyhydroxy hydrocarbon selected for the composition does notcontain an aldehyde group, the composition can additionally include adisaccharide, such as sucrose, as a stabilizer and tonicity-adjustingagent. When the composition of the present invention already contains asuitable polyhydroxy hydrocarbon (such as glycerol) and a disaccharideis employed in preferred embodiments as an additional stabilizer ortonicity-adjusting agent, the disaccharide is preferably present in arange of 5 to 25 mg/mL, more preferably 20 mg/mL, and preferably thedisaccharide is sucrose.

Pharmaceutically acceptable divalent metal salt stabilizers, such asmagnesium salts, zinc salts and calcium salts, are used in preferredembodiments of the composition of the present invention. Preferably, thesalt is a chloride salt or a magnesium salt, magnesium salt beingparticularly preferred. Preferably, the salt (e.g., the magnesium salt)is present in an amount of from about 0.1 to 1 mg/mL, more preferably inan amount of about 0.4 mg/mL.

Pharmaceutically acceptable monovalent metal salt stabilizers such aspotassium, sodium, lithium and cesium salts may be included in preferredembodiments of the present invention as optional stabilizers.Preferably, the salt is sodium chloride present in the amount of 0.6 to10.0 mg/ml, more preferably in an amount of about 5.8 mg/ml.

In addition to stabilizing the composition, sodium chloride may suppressthe rate and extent of the appearance of by-products of fermentation,resulting in a more pharmaceutically elegant presentation that may havereduced antigenicity potential due to protein aggregates. The additionof sodium chloride does not affect the pH of the formulation.

The composition of the present invention is capable of maintaining a pHin the range of about 7 to about 8.5 for extended periods of time, evenwhen subjected to harsh conditions such as refrigeration andfreeze/thaw. Moreover, the compositions can remain stable and maintainthe required pH range even when subjected to the relatively slow rate offreeze/thaw that can occur in connection with larger scale production,distribution, and handling. As noted above, maintenance of pH isimportant for viral preparations, because at pH below 7.0 and above 8.5the live virus particles can become unstable and degrade. The particularcomposition of viruses makes viruses difficult to stabilize andpreserve.

To accomplish pH maintenance under harsh conditions, the presentinvention preferably comprises a buffer system that can maintain anoptimal pH in a range from about 7.0 to about 8.5 despite storagebetween −80° C. and 27° C. and despite being subjected to freeze/thawconditions. Since pH can vary depending on temperature, pH ranges of thepresent invention are more specifically illustrated below with referenceto specific temperature ranges. For instance, at refrigerationtemperatures (e.g., about 2° C. to 8° C.) a preferred pH range is about7.7 to about 8.3, more preferably about 7.9 to about 8.2. At roomtemperature (e.g., about 20° C. to 27° C., preferably 22° C.-25° C.), apreferred pH range is about 7.3 to about 8.2, more preferably about 7.4to about 7.9.

A preferred buffer system of the present invention comprises sodiumphosphate monobasic dihydrate in a concentration of about 0.5 to 10mg/mL and tromethamine in a concentration of about 0.5 to 10 mg/mL.(Tromethamine is also known as TRIS or “Trizma” available from SigmaChemical Co.). However, other buffer systems can be used. For example,sodium phosphate dibasic dihydrate can be used if coupled with an acidicform of tris buffer. In a particularly preferred embodiment, the buffersystem comprises sodium phosphate monobasic dihydrate in a concentrationof about 1.7 mg/mL and tromethamine in a concentration of about 1.7mg/mL, and has the ability to maintain the formulation in an optimal pHrange of about 7.3 to about 7.9 at 25° C.

The formulation of the present invention has the additional advantage ofhaving the ability to stabilize high concentrations of virus at theabove-mentioned harsh conditions (such as refrigeration temperatures andfreeze/thaw processing). In particular, the formulation of the presentinvention can maintain stability of the virus at concentrations rangingup to 1×10¹³ particles/mL. A preferable range of virus concentrationsfor use in the present invention is in an amount of 1×10⁹ to 1×10¹³particles/mL., more preferably, up to 1×10¹² particles/mL, e.g. 1×10⁹(or 1×10¹⁰) to 1×10¹².

The term “diluent” as used herein can comprise a solvent (e.g., water,preferably sterile water) or a mixture of a solvent and otheringredients such as additional solvents, additional stabilizers,additional buffers, and/or other substances that do not adversely affectsafety, efficacy and stability of the formulation. With regard todiluents, stabilizers, buffers and the like, reference may be made,e.g., to Remington's Pharmaceutical Science, 15th Ed., Mack PublishingCompany, Easton, Pa.

A surfactant, preferably a nonionic detergent such as a polyoxyethylenefatty acid ester (e.g., polyoxyethylenesorbitans such as Polysorbate 20,Polysorbate 40, Polysorbate 60, or Polysorbate 80 from ICI Americas,Inc., Wilmington Del., or Tween 20, Tween, 40, Tween 60 and Tween 80from Sigma, St. Louis, Mo.), can optionally be included in thecomposition of the present invention. Preferably, the nonionic detergentis a polyoxyethylene fatty acid ester, and the polyoxyethylene fattyacid ester is preferably Polysorbate 80, which can act as a stabilizerin the composition of the present invention. The concentration ofnon-ionic detergent is preferably in a range of 0.03 to 0.3 mg/mL; morepreferably, 0.15 mg/mL.

Compositions of the present invention can further contain one or more“delivery-enhancing agents”. A “delivery-enhancing agent” refers to anyagent which enhances delivery of a therapeutic gene, such as a tumorsuppressor gene to a cancerous tissue or organ. Examples of suchdelivery-enhancing agents include but are not limited to detergents,alcohols, glycols, surfactants, bile salts, heparin antagonists,cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.

Detergents (as the term is used herein) can include anionic, cationic,zwitterionic, and nonionic detergents. Exemplary detergents include butare not limited to taurocholate, deoxycholate, taurodeoxycholate,cetylpyridium, benalkonium chloride, ZWITTERGENT® 3-14 detergent, CHAPS(3-[3-Cholamidopropyl) dimethylammoniol]-1-propanesulfonate hydrate,Aldrich), Big CHAP, Deoxy Big CHAP, TRITON®-X-100 detergent, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC®-F68 detergent, TWEEN® 20 detergent,and TWEEN® 80 detergent (CALBIOCHEM® Biochemicals).

The use of delivery-enhancing agents is described in detail in copendingU.S. patent application U.S. Ser. No. 08/889,335 filed on Jul. 8, 1997,and International Application Publication No. WO 97/25072, Jul. 17,1997, and in U.S. patent application U.S. Ser. No. 09/112,074 filed onJul. 8, 1998, International Application PCT/US 98/14241. In addition,use of calpain inhibitors in conjunction with viral vectors to increasetransduction efficiency is described in U.S. patent applications U.S.Ser. No. 09/172,685 and 60/104,321 filed on Oct. 15, 1998.

A wide range of viruses can be used in the compositions of the presentinvention, including but not limited to adenoviruses, pox viruses,iridoviruses, herpes viruses, papovaviruses, paramyxoviruses,orthomyxoviruses, retroviruses, adeno-associated virus, vaccinia virus,rotaviruses, etc. (see, e.g., Anderson, Science (1992) 256: 808-813);adenoviruses being particularly preferred. The viruses are preferablyrecombinant viruses, but can include clinical isolates, attenuatedvaccine strains, and so on. Thus, for example, an exemplary recombinantadenovirus that can be used in compositions of the invention isA/C/N/53, which is disclosed in PCT patent application no. WO 95/11984.

The formulation of the present invention is particularly well suited forstabling a recombinant virus, such as a live recombinant adenovirus (or“viral vector”), for therapeutic use in gene therapy. For instance, thevirus used in the present invention can comprise a tumor suppressorgene, such as a wild-type p53 gene or an Rb gene (e.g., p110^(RB) orp56^(RB)), and with transgenes such as wild-type p53 inserted in a viralvector, the composition of the present invention can be used as apharmaceutical composition for treatment of cancer.

In this regard, the formulations of the present invention have aremarkable ability to maintain the viability of live virus, inparticular a viral vector into which a nucleotide sequence encoding atransgene such as p53 has been inserted. This feature allows the virusto maintain its ability to infect target cells so that the therapeuticprotein encoded by the inserted transgene is adequately produced.

With specific regard to p53 and its uses, it is noted that mutation ofthe p53 gene is the most common genetic alteration in human cancers(Bartek (1991) Oncogene, 6: 1699-1703, Hollstein (1991) Science, 253:49-53). Introduction of wild-type p53 in mammalian cancer cells lackingendogenous wild-type p53 protein suppresses the neoplastic phenotype ofthose cells (see, e.g., U.S. Pat. No. 5,532,220).

In the examples below, the virus is a live recombinant adenoviruscontaining wild-type p53 gene. The particular viral vector constructused in these examples is referred to herein as “A/C/N/53”. A/C/N/53(also referred to as “ACN53”) is a particularly preferred vital vectorconstruct described in copending application U.S. Ser. No. 08/328,673filed on Oct. 25, 1994, and in WO 95/11984 (May 4, 1995), expresslyincorporated herein by reference.

A representative formula for preferred embodiments of the presentinvention that contain Polysorbate 80 is set forth below:

Representative Formula Active A/C/N/53 1 × 10⁹ to 1 × 10¹³ particles/mLSubstance Buffer Sodium 0.5 to 10 mg/mL Phosphate Monobasic Tromethamine0.5 to 10 mg/mL Stabilizer/ Sucrose 5 to 25 mg/mL tonicity agentStabilizers Glycerol 20 to 200 mg/mL Magnesium 0.1 to 1 mg/mL ChloridePolysorbate 80 0.03 to 0.3 mg/mL Solvent Water for Injection 1 mL q.s.ad(The compositions are typically stored in 1.0 mL dosages. “q.s. ad” inthe formula above means adding sufficient solvent to reach the 1 mLtotal volume).

Four particularly preferred embodiments are set forth below.(Polysorbate 80 is present in Examples 1 and 2, but absent in Examples 3and 4).

Example 1 Example 2 A/C/N/53 7.5 × 10¹¹ particles/mL 7.5 × 10¹⁰particles/mL Sodium Phosphate 1.7 mg/mL 1.7 mg/mL Monobasic DihydrateTromethamine 1.7 mg/mL 1.7 mg/mL Magnesium 0.4 mg/mL 0.4 mg/mL ChlorideHexahydrate Sucrose 20 mg/mL 20 mg/mL Polysorbate 80 0.15 mg/mL 0.15mg/mL Glycerol 100 mg/mL 100 mg/mL Water for Injection 1 mL 1 mL q.s. adpH 7.4 to 7.8 7.4 to 7.8

Example 3 Example 4 A/C/N/53 7.5 × 10¹¹ particles/mL 7.5 × 10¹⁰particles/mL Sodium Phosphate 1.7 mg/mL 1.7 mg/mL Monobasic DihydrateTromethamine 1.7 mg/mL 1.7 mg/mL Magnesium 0.4 mg/mL 0.4 mg/mL ChlorideHexahydrate Sucrose 20 mg/mL 20 mg/mL Glycerol 100 mg/mL 100 mg/mL Waterfor Injection 1 mL 1 mL q.s. ad pH 7.4 to 7.9 7.3 to 7.8

-   -   The following ingredients: sodium phosphate monobasic dihydrate,        tromethamine, magnesium chloride hexahydrate, sucrose, and        glycerol can all be obtained from, e.g., EM Industries, INC., 7        Skyline Drive, Hawthorne, N.Y. 10532. Polysorbate 80 is        available from, e.g., ICI Americas, Inc., Wilmington Del.,        19897.

Compositions of the present invention can be prepared duringpurification of the virus in a gel filtration chromatography column bycombining the ingredients (excluding Polysorbate-80) at the desiredconcentrations in the gel filtration column. (With regard to gelfiltration methods, reference can be made, e.g., to Section V below).Then, if it is desired to dilute the concentration of the virus, or toincorporate Polysorbate-80, then diluents can be prepared by standardtechniques. An illustrative example is set forth below:

Charge and dissolve sodium phosphate monobasic dihydrate, tromethamine,sucrose, magnesium chloride hexahydrate and glycerol in approximately75% of batch volume of water for injection at room temperature in astainless steel vessel equipped with agitator. Bring the batch of theresulting diluent to final volume with water for injection. Check thepH. Calculate the required volume of A/C/N/53 (adenovirus with wild-typep53 as a transgene) Drug Substance in Suspension and the required volumeof diluent to make A/C/N/53 Injection. If the final A/C/N/53 Injectionwill contain Polysorbate 80, prepare a stock solution that contains 10%excess Polysorbate 80 in diluent. Charge the calculated amounts ofA/C/N/53 Drug Substance in Suspension and diluent into a stainless steelcontainer and mix. Charge the Polysorbate 80 solution, prior to addingall of the Diluent, based upon 10% of the total A/C/N/53 Injection batchvolume if required. Aseptically filter the suspension through asterilized filter (0.22 μm or equivalent). Test the filter integrityafter filtration. Collect and fill the sterilized suspension into vialshaving the appropriate volume. Stopper and seal the vials.

Stability data for Examples 1, 2, 3, and 4 are set forth, respectively,in Tables 1, 2, 3, and 4 below.

In the Tables below, the antiproliferation assay is a bioassay used tomeasure the product's ability to suppress cancer cells and is basedgenerally on procedures used by Wills, et al., 1994, Human Gene Therapy,5:1079-1088. The numbers listed indicate activity whereas the controlhas no activity.

The “Plaque Assay” measures virus particles in culture by scoring thenumber of viral plaques as a function of dilution and is based generallyon procedures described in Graham, F. L., and Prevec, L., Methods inMolecular Biology, vol. 7: Gene Transfer and Expression Protocols, E. J.Murray, ed. (Humana Press Inc., Clifton N.J.) pp. 109-128 (1991); seealso Graham, F. L., Smiley, J., Russel, W. C., and Nairn, R., J. Gen.Virol. vol. 36, pp 59-74 (1977).

The “FACS” assay shows the ability of the virus to infect cells, andthese measurements are based generally on methods described in, e.g.,International Patent Application PCT/US97/11865 (WO 98/01582, publishedJan. 15, 1998). In the next column to the right, the numbers presentedunder the heading “Concentration” represent the concentration of thetotal number of virus particles. Finally, the numbers under the heading“Particle/FACS ratio” represent the ratio of the total number of virusparticles as compared to the number of infectious virus particles, thusindicating the relative potency of the virus preparation.

The data under the heading “UV” indicate the aggregation of the virusparticles as shown by the UV absorbance ratio for the wavelengthsA₃₂₀/A₂₆₀ as an indication of light scatter. Basically, the absorbanceat 320 nanometer wavelength measures the amount of light scatter,whereas the absorbance at 260 nanometer wavelength correlates withamount of DNA.

The temperatures listed in the second column of the tables under“condition” represent the storage temperature. The physiological assaysare performed at 37° C. and the pH in the last column is measured atroom temperature, approximately 25° C.

TABLE 1 Stability Data on Example 1 Particles Stability ConditionAntiproliferation Plaque FACS × Concentration × FACS UV Time ° C. Assay× 10⁵ SPU/mL Assay × 10⁸ PFU/mL 10¹⁰ U/mL 10¹¹ part./mL Ratio A₃₂₀/A₂₆₀pH initial 3.3 5.8 3.86 7.95 21 0.23 7.53  1 week 25 4.9 10 2.27 8.06 360.24 7.53  2 weeks 4 3.1 6.6 3.41 7.84 23 0.23 7.49  4 weeks 4 3.4 173.91 7.79 20 0.23 7.63  8 weeks 4 5.8 8.8 3.38 7.80 23 0.24 7.61 12weeks 4 3.9 36 2.24 7.80 35 0.24 7.72  5 months 4 not tested not tested1.57 8.21 52 0.26 7.60  6 months 4 3.0 7.6 2.74 7.68 28 0.28 7.58  9months 4 3.1 19 3.47 7.19* 21 0.28 7.58 11 months 4 not tested nottested nt 6.71 — 0.29 nt 12 months 4 2.6 10 0.81 6.13 76 0.30 7.62*Retest of 9 month UV samples: 6.89 × 10¹¹ particles/mL; A₃₂₀/A₂₆₀ =0.28.

TABLE 2 Stability Data on Example 2 Particles Stability ConditionAntiproliferation Plaque FACS × Concentration × FACS UV Time ° C. Assay× 10⁴ SPU/mL Assay × 10⁷ PFU/mL 10⁹ U/mL 10¹⁰ part./mL Ratio A₃₂₀/A₂₆₀pH initial 3.3 4.8 1.99 10.0  50 0.24 7.36  1 week 25 2.4 7.1 1.64 nd* —0.46 7.42  2 weeks 4 2.4 6.9 2.13 9.79 46 0.24 7.51  4 weeks 4 3.2 8.42.50 9.24 37 0.22 7.55  8 weeks 4 6.9 8.6 2.60 8.10 31 0.25 7.54 12weeks 4 5.5 8.3 1.09 8.60 79 0.24 7.64  5 months 4 not tested not tested1.74 8.03 46 0.27 7.55  6 months 4 3.3 7.3 2.10 8.25 39 0.23 7.52  9months 4 2.3 13   1.97 7.59 39 0.24** 7.53 11 months 4 not tested (nt)not tested nt 7.48 — 0.23 nt 12 months 4 1.8 9.4 0.60 4.94 82 0.26 7.59*Not determined due to assay interference. **Retest of 9 month UVsamples: 7.37 × 10¹⁰ particles/mL; A₃₂₀/A₂₆₀ = 0.24.

TABLE 3 Stability Data on Example 3 Particles Stability ConditionAntiproliferation Plaque FACS × Concentration × FACS UV Time ° C. Assay× 10⁵ SPU/mL Assay × 10⁸ PFU/mL 10¹⁰ U/mL 10¹¹ part./mL Ratio A₃₂₀/A₂₆₀pH initial 3.2 6.3 2.59 7.45 29 0.24 7.67  1 week 25 4.4 4.3 1.65 7.4945 0.24 7.68  2 weeks 4 2.3 8.0 3.62 7.27 20 0.24 7.49  4 weeks 4 2.77.6 4.08 6.97 17 0.24 7.75  8 weeks 4 5.9 8.7 2.69 7.00 26 0.25 7.82 12weeks 4 3.0 15   0.70 7.10 101  0.25 7.80  6 months 4 2.4 6.4 2.45 7.1229 0.25 7.76  9 months 4 2.8 9.4 2.51 7.06 28 0.26 7.81 11 months 4 nottested (nt) nt nt 6.71 — 0.25 nt 12 months 4 2.2 9.8 0.74 6.75 91 0.267.81

TABLE 4 Stability Data on Example 4 Particles Stability ConditionAntiproliferation Plaque FACS × Concentration × FACS UV Time ° C. Assay× 10⁴ SPU/mL Assay × 10⁷ PFU/mL 10⁹ U/mL 10¹⁰ part./mL Ratio A₃₂₀/A₂₆₀pH initial 3.3 4.7 2.36 8.91 38 0.23 7.37  1 week 25 2.3 9.3 1.40 8.2559 0.24 7.37  2 weeks 4 2.8 8.0 2.16 8.80 41 nd* 7.37  4 weeks 4 2.9 6.62.54 9.35 37 0.20 7.63  8 weeks 4 6.9 7.2 2.56 7.60 30 0.24 7.60 12weeks 4 4.4 8.6 1.45 7.20 50 0.23 7.73  6 months 4 3.6 7.1 2.85 7.92 280.21 7.58  9 months 4 2.9 11   1.87 7.26 39 0.20 7.60 11 months 4 nottested (nt) nt nt 6.93 — 0.22 nt 12 months 4 2.4 22   0.70 7.15 102 0.23 7.61 *Not determined due to assay interference.

Example 5

Formulation for Example 5: A/C/N/53 (7.5×10¹¹ Particles/mL),Tromethamine (TRIS) (1.7 mg/mL), Sodium Phosphate Monobasic Dihydrate(1.7 mg/mL), Sucrose (20 mg/mL), Magnesium Chloride Hexahydrate (0.4mg/mL), Glycerol (100 mg/mL), Sodium Chloride (5.8 mg/mL), FillVolume=10 mL.

TABLE 5 Stability Data on Example 5 Particles Stability ConditionAntiproliferation Concentration × FACS UV Time ° C. Assay × 10⁵ SPU/mLFACS × 10¹⁰ U/mL 10¹¹ part./mL Ratio A₃₂₀/A₂₆₀ pH initial 4.5 1.87 7.8142 0.23 7.80 1 month 4 8.0 1.67 7.83 47 0.23 7.80 4 month 4 13.0 1.587.84 50 0.23 7.70

In some cases, particulates have been observed to form in theformulation during storage at 4° C. Analysis of the particulates bySDS-PAGE suggests that the particulates are composed of minor impurities(i.e., additional proteins and some immature viral particles), and thusthese particulates do not affect the viability of the formulation.Nonetheless, in a preferred embodiment to further clarify theformulation (to prevent possible particulate formation), an optionalstep of microfiltration can be carried out to remove any potentialparticulates with little loss of viral particles. (When carrying outmicrofiltration, it should be noted that sufficient microfiltrationmembrane surface area per filtration volume is critical to avoid loss ofvirus as the particulate is removed.)

In addition, in a preferred embodiment, the present inventors have foundthat agitation, such as stirring, can accelerate particulate formationand is therefore an additional optional step in the clarificationprocess. Thus, gentle stirring (e.g., overnight, 10° C., using amagnetic stirbar) followed by microfiltration was shown to remove theparticulates such that no more particulate would reform upon restirring.

It was also found that cycles of freeze/thaw could promote particulateformation during restirring. Thus, in another preferred procedure, oneor more freeze/thaw cycles can optionally be carried out, followed bystirring, and then microfiltration, for the prevention of particulateformation during storage of the virus final product at refrigerationtemperatures (e.g. 4° C.).

Methods of Concentrating and Purifying Virus-Containing Compositions

The present application also discloses a new method of stablyconcentrating an existing virus preparation by employing tangential flowfiltration (hereafter sometimes referred to as “TFF”), allowing one toreadily select and prepare clinical dosages in a wide range of desiredconcentrations. The new method of concentrating a virus preparationcomprises:

-   -   (a) adding a polyhydroxy hydrocarbon to a virus preparation to a        final polyhydroxy hydrocarbon concentration of about 20% or        more; and    -   (b) subjecting the virus preparation to a filtration process        wherein the concentration of virus is increased by applying        pressure to the preparation such that diluent is removed from        the virus preparation through a filter while the virus is        retained.

The methods of the instant invention are amenable to a wide range ofviruses, including but not limited to adenoviruses, pox viruses,iridoviruses, herpes viruses, papovaviruses, paramyxoviruses,orthomyxoviruses, retroviruses, adeno-associated virus, vaccinia virus,rotaviruses, etc.; adenoviruses being particularly preferred. Theviruses are preferably recombinant viruses, but can include clinicalisolates, attenuated vaccine strains, and so on. The present inventionis particularly useful for concentrating recombinant viruses carrying aheterologous transgene for use in gene therapy. Such viruses areespecially vulnerable to potentially destabilizing forces, such as theadditional shear mechanical forces accompanying methods of concentratingvirus preparations. An exemplary recombinant adenovirus that can beconcentrated by the method of the invention is A/C/N/53, which isdisclosed in PCT Patent Application No. WO 95/11984.

The filtration process used to concentrate the virus according to themethod of the present invention can include any filtration process(e.g., ultrafiltration) where the concentration of virus is increased byforcing diluent to be passed through a filter in such a manner that thediluent is removed from the virus preparation whereas the virus isunable to pass through the filter and thereby remains, in concentratedform, in the virus preparation. Ultrafiltration is described in detailin, e.g., Microfiltration and Ultrafiltration: Principles andApplications, L. Zeman and A. Zydney (Marcel Dekkar, Inc., New York,N.Y., 1996). A particularly preferred filtration process is TangentialFlow Filtration (“TFF”) as described in, e.g., MILLEPORE catalogueentitled “Pharmaceutical Process Filtration Catalogue” pp. 177-202(Bedford, Mass., 1995/96). Preferred TFF apparatus comprises either aPellicon II or Pellicon XL filter system from Millipore Corporation, 80Ashby Rd., Bedford, Mass. (internet address: www.millipore.com), aPellicon XL system being particularly preferred. In a preferredembodiment, the methods of the present invention are carried out attemperatures in a range from about 2° C. to 27° C.

Other concentration processes can be employed to concentrate viruspreparations in accordance with the present invention. For instance,employment of polyhydroxy hydrocarbon can advantageously be used toconcentrate a virus preparation by centrifugation. Thus, the presentinvention also provides a method for concentrating a virus preparationcomprising:

-   -   (a) centrifuging a composition which comprises a first layer        comprising a polyhydroxy hydrocarbon in a concentration of 35%        to 80% (v/v), the first layer overlaid with a second layer        comprising a polyhydroxy hydrocarbon in a concentration of 5% to        30% (v/v), the second layer overlaid with a third layer        comprising virus; and    -   (b) recovering the virus from the first layer.        By way of example, an adenovirus preparation can be concentrated        by low speed centrifugation at 3,200 g using swing bucket rotors        of a Beckman centrifuge. To accomplish this, the virus        preparation can be placed into multiple 5 ml tubes, each tube        containing 6.25% volume of 70% glycerol in a first layer at the        tube bottom, overlaid with 2.5% volume of 20% glycerol with the        virus preparation laid on top. The preparation is then        centrifuged at 3,200 g at 4° C. for approximately 16 hours to        pellet the concentrated virus into the glycerol layers and then        the newly-concentrated virus preparation is subsequently        recovered from the first layer. Virus concentrated by the        procedures described above had good light scattering        characteristics and had suitable infectivity properties.

With regard to the polyhydroxy hydrocarbon used in the methods of thepresent invention, a “polyhydroxy hydrocarbon” means a branched, linear,or cyclic compound substituted with 2 or more (preferably 2 to 6, morepreferably 2 to 4) hydroxy groups, and an effective amount ofpolyhydroxy hydrocarbon is an amount sufficient to stabilize the virusagainst potentially destabilizing forces, such as the mechanical shearforces that occur during the concentration process. Preferably, thepolyhydroxy hydrocarbon in the virus-concentrating methods of thepresent invention is present in a minimum concentration of 20%, morepreferably 25%. Polyhydroxy hydrocarbons for use in the presentinvention preferably are polyhydroxy-substituted alkyl compounds(branched or unbranched), preferably having 2 to 7 carbon atoms, and caninclude glycerol, sorbitol and polypropanol. Glycerol is particularlypreferred.

The inventors' new method of increasing virus concentration has theadditional advantage of enhancing processing, e.g., by eliminatingproblematic bottlenecks by allowing significantly higher throughputduring various processing steps such as size exclusion chromatography.Thus, in a preferred: embodiment, the method of concentrating viruspreparations in accordance with present invention can be applied tomethods of purifying viruses where a size exclusion chromatography step(e.g., gel filtration) is performed subsequent to anion exchangechromatography. In this embodiment, there are additional threats tovirus stability stemming not only from the mechanical shear forcesneeded to concentrate the virus prior to the rate-limiting sizeexclusion chromatography step, but also due to the fact that the viruspreparation eluted from the anion exchange chromatography step typicallycontains high levels of salts and other impurities that furthercompromise virus stability. Thus, in a particularly preferredembodiment, the present invention provides a method of purifying a viruspreparation comprising:

-   -   (a) subjecting the virus preparation to anion-exchange        chromatography, wherein the virus is eluted as a virus        preparation product from an anion-exchange chromatographic        medium;    -   (b) adding a polyhydroxy hydrocarbon to the virus preparation        product of step (a) so that the concentration of polyhydroxy        hydrocarbon in the preparation reaches a final concentration of        about 25% or more; and    -   (c) increasing the concentration of virus in the virus        preparation product of step (b) by applying pressure to the        preparation such that diluent is removed from the virus        preparation through a filter while the virus is retained; and    -   (d) subjecting the concentrated virus preparation product of        step (c) to one or more additional processing steps.

In the preferred embodiment set forth above in connection with anionexchange chromatography, the minimum level of glycerol is 25% (ratherthan the 20% minimum level in general applications of the concentrationmethods of the present invention) because this particular applicationmust take into account the additional threat to stability posed by thehigh salt concentrations in the product eluted from the anion exchangecolumn. The addition of 25% glycerol (preferably 30%) results instability of the salt-containing DEAE pool for >10 days at, e.g., 4° C.;therefore subsequent steps of virus concentration and/or gel filtrationcan be performed on separate days with substantial flexibility across a10 day period. As will be appreciated, the employment of polyhydroxyhydrocarbon in the higher concentration of 25% or more can also be usedin methods of the present invention when the virus preparation containshigh salt content due to other processing conditions.

V. EXAMPLES OF METHODS OF THE PRESENT INVENTION

The following examples illustrate preferred embodiments of the presentinvention; the scope of the invention is not to be construed as limitedthereby.

Brief Overview—A concentrated batch starts with frozen crude viralmaterials originating from fermentation recovery. In one embodiment, theadenovirus product is first purified by anion exchange chromatography.Then, prior to loading the preparation onto a size exclusion column, theanion exchange pool can be concentrated by tangential flow filtration(TFF) in the presence of 30% (v/v) glycerol. Alternatively, in anotherembodiment, the TFF concentration step can be carried out in thepresence of 20% (v/v) or more (preferably 25%) glycerol after sizeexclusion chromatography.

Preparation of Starting Materials by Anion-Exchange Chromatography Priorto TFF

In a preferred embodiment, an adenovirus anion exchange pool is preparedfor concentration as follows. Frozen viral material from fermentationand recovery steps is thawed and filtered through a 0.45 μm hydrophilicmembrane. The salt concentration of the filtrate is adjusted by adding4M sodium chloride. This feed solution is then applied to a FractogelEMD DEAE-650M column pre-equilibrated with 50 mM sodium phosphate pH7.5, 260 mM sodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose(Buffer A). The adenovirus binds to the anion exchange resin, whereasthe majority of media and host cell impurities pass through the columnending up in the spent charge. The column is initially washed with 4volumes of buffer A followed by a second isocratic wash of 8 bed volumesof 94% buffer A and 6% buffer B (50 mM sodium phosphate pH 7.5, 600 mMsodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose) to removeadditional impurities. The virus is eluted from the column with a 30 bedvolume linear gradient from 6% to 100% buffer B. The Adenovirus peak ofthe elution profile as determined by A₂₈₀ is collected. Then glycerol isadded to the DEAE pool at a final concentration of 30% (v/v) for furtherprocessing.

Concentration of DEAE Pool Using Tangential Flow Filtration

The DEAE pool (prepared in accordance with the above description) isconcentrated to 10- to 20-fold by using a Millipore TFF unit (PelliconXL System) with 1 million molecular weight cut-off Biomax membranes. Theprocess is carried out either at 2-10° C. or room temperature (25° C.).The following filtration parameters are used in this procedure: averageinlet pressure=14 psi; average permeate pressure=o psi; average fluxrate=13 liters/hour-square meter. The final concentration of adenovirusachieves approximately 1.0−2.0×10¹³ particles per ml. Based on theResource Q-HPLC and UV absorbance (A₂₆₀) analysis, the recovery ofconcentration step is >80% with no significant aggregation (lightscattering assay by A₃₂₀/A₂₆₀).

Buffer Exchange by Size Exclusion Chromatography (Gel Filtration)

The concentrated adenovirus preparation is applied to a Superdex-200size exclusion column pre-equilibrated with 20 mM sodium phosphate pH8.0, 100 mM sodium chloride, 2 mM magnesium chloride, 2% (w/v) sucrose,10% glycerol (Buffer C) or 11 mM sodium phosphate, 14 mM Tris, 2 mMmagnesium chloride, 2% (w/v) sucrose, 10% glycerol, pH 7.8 (Buffer D).The column is eluted with equilibration buffer. The Adenovirus peak ofthe elution profile as determined by A₂₈₀ is collected and pooled. Theconcentrated adenovirus preparation is filtered through a 0.2 μmhydrophilic Durapore membrane (Stericup, Millipore) at 2 to 10° C., andcan be stored at −80° C., or at higher temperatures (such as 2 to 10°C.).

Concentration of Superdex-200 Pool Using Tangential Flow Filtration

As discussed above, a preferred embodiment of the present inventioninvolves concentrating the virus after anion exchange chromatography,but before gel filtration. However, in another embodiment, the disclosedmethods of concentrating virus preparations can also be used after thegel filtration step (even if no virus concentration step was employed inbetween the anion exchange step and the gel filtration step). In thiscase, the filtration parameters are the same as those for concentrationof a DEAE pool, except that the polyhydroxy hydrocarbon (e.g., glycerol)can be added to the Superdex-200 pool at a final concentration as low as20% (v/v) since it is no longer necessary to deal with the high saltconcentrations in the DEAE pool. In this regard, it should be noted thatin cases where the addition of polyhydroxy hydrocarbon is postponeduntil after gel filtration, the DEAE pool should be applied immediatelyto the gel filtration column (due to the vulnerability of the DEAEpool—with its high salt concentration). Thus, it can be seen that anadditional advantage of adding polyhydroxy hydrocarbon to the DEAE pool(in accordance with the present invention) is increased flexibility interms of the time interval and storage options during the period of timebetween anion-exchange chromatography and subsequent processing.

The methods of concentrating virus preparations can be applied inconnection with a variety of purification methods. For additionalinformation on purification methods, reference can be made, e.g., toHuyghe et al., Human Gene Therapy, Vol. 6, pp. 1403-1416 (1995) and U.S.patent application Ser. No. 08/989,227, expressly incorporated herein byreference.

VI. STABILITY DATA FOR METHODS OF CONCENTRATING VIRUS PREPARATIONS USINGTANGENTIAL FLOW FILTRATION

As shown by the experimental data below, the methods of the presentinvention allow for greatly enhanced virus stability, despite themechanical shear forces of concentrating the virus, and despite harshconditions such as high salt levels in a DEAE pool. Thus, methods of thepresent invention allow for, inter alia, (1) ready preparation ofclinical dosages at any desired concentration (even when starting withmaterial having a lower concentration), (2) enhancement of processing(e.g., by allowing significantly higher throughput during size exclusionchromatography), and (3) stability of the salt-containing DEAE poolfor >10 day at 2-10° C. (thus allowing for subsequent steps of virusconcentration and/or gel filtration to be performed on separate dayswith substantial flexibility across a 10 day period.

A. Concentrating Virus Subsequent to DEAE Chromatography

In the following three examples, stable concentrations of adenoviruswere prepared by concentrating DEAE Pools in 30% glycerol (in accordancewith the methods of the present invention). The preparations were thensubjected to further purification by Superdex-200 gel filtrationchromography to obtain the final formulation for testing.

Example D-1 Final Formulation:

-   -   20 mM NaPi, 100 mM NaCl, 2 mM MgCl₂, 2% sucrose, 10% glycerol,        pH 8 at 2-10° C.

Results:

-   -   Particles/FACS=24    -   Light Scattering (A320/A260)=0.22    -   Conc.=1.6×10¹³ particles/ml

Example D-2 Final Formulation:

-   -   14 mM Tris base 11 mM NaPi, 2 mM MgCl₂, 2% sucrose, 10%        glycerol, pH 7.8 at 2-10° C.

Results:

-   -   Particles/FACS=17    -   Light Scattering (A320/A260)=0.25    -   Conc.=1.5×10 ¹³ particles/ml

Example D-3 Final Formulation:

-   -   20 mM NaPi, 100 mM NaCl, 2 mM MgCl₂, 2% sucrose, 10% glycerol,        pH 8 at 2-10° C.

Results:

-   -   Particles/FACS=24    -   Light Scattering (A320/A260)=0.25    -   Conc.=1.3×10¹³ particles/ml.

B. Concentrating Virus Subsequent to Gel Filtration Example S-1

In the following example, the virus preparation was concentrated in 20%glycerol subsequent to gel filtration.

Final Formulation:

-   -   16 mM NaPi, 80 mM NaCl, 1.6 mM MgCl₂, 1.6% sucrose, 20%        glycerol, pH 8 at 2-10° C.

Results:

-   -   Particles/FACS=72;    -   Light Scattering (A320/A260)=0.26    -   Conc.=1.66×10¹³ particles/ml

All publications, patents and patent applications cited herein areincorporated in their entirety by reference to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

Modifications and variations of this invention will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is not to be construedas limited thereby.

1-28. (canceled)
 29. A purified adenovirus composition obtained by amethod of purifying a virus preparation, the method comprising the stepsof: (a) subjecting the virus preparation to anion-exchangechromatography, wherein the adenovirus is eluted from an anion-exchangechromatographic medium; and (b) subjecting the elute from step (a)containing the adenovirus to size exclusion chromatography, wherein theadenovirus is eluted from a size exclusion chromatographic medium. 30.The adenovirus composition of claim 29, wherein the virus preparation isa cell lysate.
 31. The adenovirus composition of claim 30, wherein thecell lysate is filtered before step (a).
 32. The adenovirus compositionof claim 30, wherein the cell lysate is subject to nuclease treatmentbefore step (a).
 33. The adenovirus composition of claim 29, wherein theadenovirus is recombinant.
 34. The adenovirus composition of claim 29,wherein the anion-exchange medium is DEAE-FRACTOGEL.
 35. The adenoviruscomposition of claim 29, wherein the size exclusion medium isSUPERDEX-200.
 36. The adenovirus composition of claim 29, wherein theanion-exchange medium comprises diethylaminoethyl groups on across-linked agarose, cellulose, polyacrylamide or polystyrene backbone.37. The adenovirus composition of claim 29, wherein the size-exclusionmedium comprises a cross-linked polysaccharide.
 38. The adenoviruscomposition of claim 37, wherein the cross-linked polysaccharide is acomposite of cross-linked agarose and dextran.
 39. The adenoviruscomposition of claim 29, wherein the anion-exchange chromatographicmedium is extensively washed before step (a).
 40. The adenoviruscomposition of claim 29, wherein step (b) further comprises eluting theadenovirus from the size-exclusion chromatographic medium into alow-salt buffer by a high-salt elution buffer, wherein thesize-exclusion medium is in a column containing a salt gradient whichdecreases in ionic strength from the top of the column towards thebottom of the column.
 41. The adenovirus composition of claim 29,wherein the anion-exchange chromatographic medium or the size exclusionchromatographic medium is contacted with a buffer comprising glycerol,including wash, equilibration, loading and elution buffer.
 42. Theadenovirus composition of claim 29, wherein step (a) is performed by:(i) pre-equilibrating a DEAE-EMD FRACTOGEL 650M column with 5 bed volume(BV) of 0.5 M NaOH/1M NaCl, by 6 BV of 0.1 M HCl/1 M NaCl, and 20 BV ofBuffer A, wherein Buffer A is a solution consisting of 265 mM NaCl, 2 mMMgCl₂, 2% (w/v) sucrose, and 50 mM sodium phosphate at pH 7.5; (ii)loading the virus preparation to the column and subsequently washing thecolumn with 4 BV of Buffer A, wherein the virus preparation prior toloading has a conductivity equal to that of Buffer A; (iii) washing thecolumn with 8 BV of 94% Buffer A/6% Buffer B, wherein Buffer B is asolution consisting of 600 mM NaCl, 2 mM MgCl₂, 2% (w/v) sucrose, and 50mM sodium phosphate at pH 7.5; and (iv) eluting the adenovirus with 30BV of a linear gradient from 94% Buffer A/6% Buffer B to 100% Buffer B.43. The adenovirus composition of claim 29, wherein step (b) isperformed by: (i) pre-equilibrating a SUPERDEX-200 column with 0.5 bedvolume (BV) of 0.5 M NaOH, 1 BV of H₂O, and 2 BV of Buffer C, whereinBuffer C is a solution consisting of 130 mM NaCl, 2 mM MgCl₂, 2% (w/v)sucrose, and 50 mM sodium phosphate at pH 7.5; (ii) loading the elutefrom step (a) to the column; and (iii) eluting the adenovirus withBuffer C.
 44. The adenovirus composition of claim 29, wherein step (b)is performed by: (i) pre-equilibrating a SUPERDEX-200 column with 0.5bed volume (BV) of 0.5 M NaOH, 1 BV of H₂O, and 2 BV of Buffer C,wherein Buffer C is a solution consisting of 130 mM NaCl, 2 mM MgCl₂, 2%(w/v) sucrose, and 20 mM sodium phosphate at pH 8.0; (ii) loading to thecolumn 0.15 BV of a linear gradient from 100% Buffer C to 100% Buffer D,wherein Buffer D is a solution consisting of 420 mM NaCl, 2 mM MgCl₂, 2%(w/v) sucrose, and 20 mM sodium phosphate at pH 8.0; (iii) loading theelute from step (a) to the column; and (iv) eluting the adenovirus withBuffer D.
 45. The adenovirus composition of claim 29, wherein step (a)is performed by: (i) pre-equilibrating a FRACTOGEL EMD DEAE-650M columnwith Buffer J-1, wherein Buffer J-1 is a solution consisting of 265 mMNaCl, 2 mM MgCl₂, 2% (w/v) sucrose, and 50 mM sodium phosphate at pH7.5; (ii) loading the virus preparation to the column; (iii) washing thecolumn with 8 BV of 94% Buffer J-1/6% Buffer J-2, wherein Buffer J-2 isa solution consisting of 600 mM NaCl, 2 mM MgCl₂, 2% (w/v) sucrose, and50 mM sodium phosphate at pH 7.5; and (iv) eluting the adenovirus with30 BV of a linear gradient from 94% Buffer J-1/6% Buffer J-2 to 100%Buffer J-2.
 46. The adenovirus composition of claim 29, wherein step (b)is performed by: (i) pre-equilibrating a SUPERDEX-200 column with BufferK-1, wherein Buffer K−1 is a solution consisting of 100 mM NaCl, 2 mMMgCl₂, 2% (w/v) sucrose, and 20 mM sodium phosphate at pH 8.0; (ii)loading the elute from step (a) to the column; and (iii) eluting theadenovirus with Buffer K-1.
 47. The adenovirus composition of claim 46,wherein the elute from step (iii) is subsequently filtered through a 0.2μm hydrophilic DURAPORE membrane at 2-15° C.